Protein glycosylation is a common post-translational modification in all animals that creates post-genomic diversity, but systems-level approaches for glycomics are lacking in most biological settings. A primary need is simple and sensitive technology that can be used to analyze multiple glycans synthesized by cells (the cellular glycome). This type of analysis has been challenging because of the diversity and complexity of glycans, the low abundance of certain glycan species, the poor sensitivity of existing glycomics approaches, and the lack of efficient and unbiased strategies for releasing glycans from complex samples. Current technologies for evaluating glycans require relatively large amounts of biological samples for detailed structural analyses, which limits their widespread application.
One of the most common types of protein glycosylation is mucin-type O-glycosylation (R-GalNAc-α-O-Ser or -Thr), where R represents extended structures, which is present on the majority of proteins that traverse the secretory apparatus and is important in many normal and pathologic settings. Nonetheless, little is known about either the repertoire of O-glycans or how specific O-glycan structures regulate biology, largely because of a lack of effective technologies for O-glycomics. In contrast to N-glycans, which can be released enzymatically, O-glycans require chemical strategies for their release—primarily alkaline β-elimination, which is inefficient, is potentially biased, and may result in O-glycan degradation via peeling reaction. Thus, there is a need to identify improved methods for evaluating O-glycans in living cells.
Brockhausen et al report in vitro N-acetylglucosaminyltransferase (GlcNAc-transferase) activities using synthetic oligosaccharide alpha-O-glycosides, i.e., GalNAc-α-O—R, as GlcNAc substrates, wherein R is phenyl (Ph), benzyl (Bn), or o-nitrophenyl (ONP) instead of a peptide. Carbohydrate Research, 1983, 120, 3-16. Sarkar et al. report the inhibition of sialyl Lewis X by acetylated Gal(beta)1-4GlcNAc(beta)-O-naphthalenemethanol. Proc. Natl. Acad. Sci. USA, 1995, 92:3323-3327.
Kuan et al. report that aryl-N-acetyl-alpha-galactosaminides can be useful for the structural and functional studies of mucin macromolecules and other O-linked glycoproteins. J Biol Chem, 1989, 264(32):19271-7. See also Delannoy et al. Benzyl-N-acetyl-alpha-D-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation. Glycoconj. J. 13, 717-726 (1996) and Zanetta, et al. Massive in vitro synthesis of tagged oligosaccharides in 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside treated HT-29 cells. Glycobiology 10, 565-575 (2000).
Kudelka et al. report cellular O-glycome reporter/amplification to explore O-glycans of living cells. Nat Methods, 2016, 13(1):81-6.
References cited herein are not an admission of prior art.